Although the immune responses via toll-like receptors (TLRs) protect cells from virulent damages, if left unchecked, pathogen-stimulated excessive production of cytokines can lead to organ failures and various severe inflammatory diseases. Our long-term goal is to elucidate the signal mechanisms underlying TLR recognizing specific pathogen products and meanwhile controlling cytokine production through both agonist- specific and synergistic TLR signaling pathway(s). Base on our findings that in the LPS-stimulated macrophages a novel TLR4-interacting protein functions as a differential regulator of NFkappaB activation, we hypothesize that many TLR downstream proteins of unknown function differentially regulate the switching on &off of TLR signaling in a cooperative and timely manner. By using our newly integrated systemic technology platform, we have shown that these novel signal proteins can be systematically identified characterized in a TLR-mediated pathway in actual immune cells in real-time. To resolve the complexity of the disease pathogenesis-associated signal protein interaction networks (interactomes), we plan to: (1) Conduct the 'pathway-scale'profiling of the multiprotein signal complexes along agonist-specific TLR signal transduction relays. Our 'dual-tagging'profiling integrates the capabilities of natural complex formation, epitope affinity isolation, and 'in-spectra'quantitative measurements required for system-scale investigation that will be performed for comprehensive analyses of 'dual-tagged'bait-containing complexes isolated from various agonist-stimulated living macrophages, (2) Perform a 'pathway-scale'characterization of the functional roles/links of novel signal proteins in signal modulation in agonist-specific TLR- mediated pathways. The function and mechanistic characterization of novel proteins/their interactions will shed new light on selective regulation of TLR-mediated signals by their downstream interactome recruited following agonist-specific stimulations, and (3) Characterize the multi-TLR-mediated signaling interactome correlated to disease pathogenesis-related TLR synergy. Pathogens may contain several TLR agonists that trigger multiple TLR-mediated signaling pathways, synergistically contributing to disease pathogenesis through improper production of inflammatory cytokines. Our systemic approach has a unique strength to resolve the complexity of the signaling interactome in operating multi-pathway coordination and 'cross- talking'. Taken together, the molecular understanding of the signal interactomes responsible for selective signal modulation will provide a new insight into the control of human inflammatory diseases.